X chromosome inactivation is a form of chromosome-wide gene regulation which results in the silencing of one of the two X chromosomes in female mammals, thereby restoring equal dosage of gene expression between males and females. There are significant differences in this inactivation process between mammal groups, which probably reflects its evolution. One of the most striking difference is that in metatherian mammals (marsupials) only the paternal X chromosome is inactivated, whereas eutherian (placental) mammals show random inactivation in most tissues. X inactivation is initiated at the XIST locus in eutherians, but no homologous locus has yet been demonstrated in marsupials. X inactivation is less stable in marsupials than in eutherians, and is incomplete and tissue-specific. The molecular basis, too may be different. Although differences in histone acetylation have been demonstrated between active and inactive X chromosomes in marsupials as well as eutherians, DNA methylation of the promoter region of inactive genes have not, perhaps accounting for the less stable inactivation in this mammalian group. These differences between the X inactivation processes in marsupials and eutherian mammals suggest that marsupials may have retained a less complex and more primitive form of X inactivation. The differences between the groups offer a unique opportunity to dissect out the layers of the process of gene silencing at a chromosome level. Our goal is to investigate these differences by a series of molecular approaches. We plan first to isolate the XIST locus from marsupials, based on our preliminary finding of a female-specific RNA signal using a probe enriched in marsupial X chromosome DNA. We will compare eutherian and metatherian XIST sequence and methylation status to examine possible XIST imprinting in marsupials. Secondly, we will investigate the stability of X inactivation of marsupial genes in comparison to mouse by measuring expression from the active and inactive X chromosomes in individual cells. Thirdly, we will insert a marsupial gene into the mouse X chromosome to determine whether the gene is susceptible or resistant in vivo to epigenetic changes which characterize eutherian genes.